Figure 4. Both ACLY and ACSS1 are involved in α₂M^*/CS-GRP78-induced acetylation of proteins.

(A) Immunoblot analysis of indicated proteins in DU145-shACSS1 cells treated with C38 Mab (50 μg) for 6 h and then stimulated with α₂M^* (100 pM) for 30 min and acetate (5 mM) for 4 h. (B-C) ACSS1 silencing of DU145 cells were stimulated either alone or in combination with α₂M^* (100 pM) for 30 min or acetate (5 mM) for 4 h in the absence and presence of ACLYi (100 μM) for 16 h, AKTi (GSK690693, 5 μM/L) for 16 h or C38 Mab (50 μg) for 6 h to quantify the transcript level of ACSS1 and ACLY genes. (D) The total cellular concentration of acetyl-CoA was measured from ACSS1 silencing of DU145 cells stimulated either alone or in combination with α₂M^* (100 pM) for 30 min or acetate (5 mM) for 4 h in the absence and presence of ACLYi (100 μM) for 16 h, AKTi (GSK690693, 5 μM/L) for 16 h or C38 Mab (50 μg) for 6 h. mean ± SEM of triplicates. (E) Immunoblot analysis of histone acetylation levels in ACSS1 silencing of DU145 cells were stimulated either alone or in combination with α₂M^* (100 pM) for 30 min or acetate (5 mM) for 4 h in the absence and presence of ACLYi (100 μM) for 16 h, AKTi (GSK690693, 5 μM/L) for 16 h or C38 Mab (50 μg) for 6 h. (F) Immunoblot analysis of protein acetylation in the indicated cell lines stimulated either alone or in combination with α₂M^* (100 pM) for 30 min or acetate (5 mM) for 4 h in the absence and presence of ACLYi (100 μM) for 16 h, AKTi (GSK690693, 5 μM/L) for 16 h or C38 Mab (50 μg) for 6 h. (G) Immunoblot analysis of protein acetylation levels in ACLY silencing of indicated cells were treated with C38 Mab (50 μg) for 6 h and then stimulated with α₂M^* (100 pM) for 30 min or acetate (5mM) for 4 h either alone or in combination. (H) Immunoblot analysis of protein acetylation levels in ACSS1 silencing of DU145 cells were stimulated either alone or in combination with α₂M^* (100 pM) for 30 min or acetate (5mM) for 4 h in the absence and presence of ACLYi (100 μM) for 16 h, AKTi (GSK690693, 5 μM/L) for 16 h or C38 Mab (50 μg) for 6 h. ^*, p values ≤ 0.05. Error bar represent S.D.